PROJECT SUMMARY Mitochondrial integrity and function are critical to neurons, which rely on oxidative phosphorylation to meet their high-energy demands. The failure to maintain mitochondrial health plays a central role in multiple neurological disorders, including ischemic stroke, Parkinson's Disease, and Alzheimer's Disease. Mitochondria are regulated through fusion, fission, transport, biogenesis, mitophagy, and multiple stress response pathways. While these processes have been well studied in tissue culture models, we do not have a full understanding of how they are mediated in specific tissues (particularly neurons) in vivo or how they are interdependent. Importantly, we do not know the identity of all of the molecular players that mediate these processes. To address these questions, we have used the genetic model system C. elegans to perform a novel unbiased forward genetic screen for mutants with altered mitochondrial morphology, number, or transport. Here we propose to use whole genome sequencing to identify the underlying genes, as their identity will supply missing pieces to the molecular mechanisms that mediate mitochondrial biology. We will then identify their mammalian orthologs and characterize their function with respect to mitochondria in cultured mouse cortical neurons by either overexpressing wild-type protein or knocking down endogenous protein using small hairpin RNAs. Forward genetic screens fit the criteria of ?high risk, high reward? often employed in NIH R21 applications. The risk is that the screen is unbiased and therefore not hypothesis-driven; we have no idea what genes we will identify. But the unbiased nature allows us to identify factors that otherwise would have escaped attention by traditional hypothesis-based approach, which is the reward. Our proposed collaborative experiments will determine which genes play a conserved role in mitochondrial function and thus direct the focus of our long- term efforts. Combined, these experiments will found a new collaboration between the Rongo and Cai labs, allowing us to generate compelling preliminary data for a hypothesis-driven NIH R01 application. The goal of such a future R01 application will be to study the molecular and cell biological mechanism of several of these conserved factors in mitochondrial cell biology in neurons using C. elegans in vivo and mammalian tissue culture in vitro complementary approaches.